Method for the production of N-acylpentapeptides

ABSTRACT

An improved method for the fermentative production of Nacylpentapeptides by cultivation of Streptomyces naniwaensis (ATCC 21689), a kind of Actinomyces, is provided wherein a nutrient medium is employed for the cultivation of said Actinomyces, said nutrient medium additionally contains a precursor of N-acylpentapeptide; an increased yield of the desired N-acylpentapeptide being thus obtained.

United States Patent 1 Kuwana et al.

11 3,929,572 Dec. 30, 1975 METHOD FOR THE PRODUCTION OF N-ACYLPENTAPEPTIDES [75] Inventors: Noriaki Kuwana, Inuyama; Kouichi Suzuki, lchinomiya; Tooru Sugitani, Aichi, all of Japan [73] Assignee: Eisai Co., Ltd., Tokyo, Japan [22] Filed: Nov. 8, 1974 [21] Appl. No.: 522,268

[30] Foreign Application Priority Data Nov. 15,1973 Japan 48-127716 [52] U.S. Cl 195/29; l95/30 [51] Int. Cl. C12D 13/06 [58] Field of Search 195/30, 29, 80 R [56] References Cited OTHER PUBLICATIONS Murao et al., Agr. Biol. Chem., Vol. 35, No. 10, pp.

Primary Examiner-Alvin E. Tanenholtz Attorney, Agent, or FirmWenderoth, Lind & Ponack 57 ABSTRACT 4 Claims, No Drawings furthermore, a particular N-acylpentapeptide can be METHOD FOR THE PRODUCTION OF obtained predominantly when a specific precursor cor- N-ACYLPENTAPEPTIDES responding'to the acyl moiety of said particular N-acylpentapeptide is added. to the nutrient medium to be This invention relates to an improved method for the 5 used.

production of N-acylpentapeptides. More particularly, The term precursor" herein used means the subthe invention is concerned with a new method for the stances which will provide a moiety to result in a correproduction of N-acylpentapeptides in a high yield by sponding N-acylpentapeptide including the substances, cultivating Streptomyces naniwaensis in a nutrient mewhich may hereinafter be called N-acyl radical dodium containing a precursor or precursors of said N- nor, exemplified by 4-amino-3-hydroxy-6-methylacylpentapeptides. heptanoic acid (AHMl-IA') per se or in a form of its N-acylpentapeptides obtained in accordance with the derivatives, for example. present invention are represented by the following As for the N-acyl radical donors, there may be mengeneral formula: V tioned aliphatic acids in a form of their inorganic salts,

CH3 CH3 CH H CH CH CH cH H 0H, OH

wherein R is acyl radical. esters and amides; such as sodium, potassium and am- The N-acylpentapeptides of the above formula (I), monium salts of acetic, propionic and butylic acids; wherein R is butyl, propyl or acetyl, are disclosed by N. such as the esters derived from a higher alcohol, polyal- Kuwana et al in Japanese Patent Application Nos. cohol and other alcohols and an aliphatic acid, for 39446/73 and 68310/73 as Pepsidines A, B and C. example, stearyland oleyl acetates, propionates and In Japanese Patent Application No. 35900/70, said butylates; and such as the amides derived from an Pepsidine C is represented by S. Murao et al as S-PI. amino radical-containing compounds, for example, an Other N-acylpentapeptides of the above formula (I) aminoacid and aminosugars, especially, S-acetylvaline having other acyl radicals are known, too. and N-acetylglucosamin. In this connection, it has been It is known that all of these N-acylpentapeptides have found that use of a co-enzyme-type acetyl radical a high anti-pepsin activity and they are the products donor such as thiol esters of the fatty acids exemplified obtained by cultivation of various Actinomyceses. by S-acetyl glutathion is particularly preferable for the Thus, the aforementioned Pepsidines A, B and C are predominant production of Pepsidine C.

the products of the cultivation of Streptomyces naniw- The amount of the precursor(s) to be used is not aensis EF 44-201 (ATCC 21689), a kind of Ancritical. It is, however, usually preferable to use in an tinomyceses, for example. amount of ODS-2.0% by w/v of a single precursor or an For the cultivation of Actinomyces to produce the admixture thereof on the basis of the employed nutri- N-acylpentapeptides, there may generally be employed ent medium in order to obtain a desired result accordvarious nutrient mediums which contain several ingreing to the present invention.

dients conventionally employed in the preparation of The precursor or precursors may be incorporated the nutrient mediums to be used for the cultivation of 45 into a nutrient medium in a form of its aqueous soluother Actinomyceses. tion. They may thus be added at once or intermittently Thus, a nutrient medium, for example, may contain to the medium.

sugars such as glucose and the like as the carbon The compositions of the nutrient medium for the source; peptone and meat extract, for example, as the concomitant use with the pre cursos(s) and the condinitrogen source;and sodium chloride and various other tions employed for the cultivation of Actinomyces inorganic salts. naniwaensis EF 44-201 strain in said medium accord- It is known that the yield of N-acylpentapeptides is ing to the present invention are not critical. Usually, affected by the kinds and/or relative proportions of the cultivation may be carried our under shaking andthese ingredients contained in a given nutrient medium. /or aerating at a temperature of about 20C. to about [See Japanese Agricultural and Biological Chemistry 24C. and for 10 to 120 hours.

35, No. 10, 1477-1481 (1971) and Japanese Patent The amounts of the Pepsidines produced in the culti- Publication No. 8996/72.] vated broth are determined in accordance with the It has, however, been found that a production of improved procedure for the determination of anti-pep- N-acylpentapeptides in a gOOd yield and/or a selective sin activity disclosed in Japanese Patent Application production of a particular N-acylpentapeptide among No. 35900/70. Namely, 0.5 ml ofa sample taken from others could not be attained by the use of a nutrient the cultivated broth is diluted with a buffer solution to medium consisting merely of the usual ingredients for a suitable concentration, and there is added 0.5 ml ofa the cultivation of Actinomyces. 0.01% pepsin solution. The resulting mixture is warmed The present inventors now have found that the yield at 37C. for 10 minutes. To the mixture, there is added of N-acylpentapeptides can considerably be increased 2.5 ml of a 1% aqueous casein solution (pH 1.6), when a precursor or precursors of the N-acylpentapepwarmed at 37C. for 10 minutes and finally added 2 ml tide is/are incorporated into a conventional nutrient of a 0.55 M trichloroacetic acid. The mixture is held in medium used for the cultivation of Actinomyces, and a boiling water bath for 10 minutes and then ice- 4 that addition of the aqueous sodium acetate solution was eliminated.

The resulting N-acylpentapeptides contained in the filtrate was estimated according to the method aforementioned. The results thus obtained are shown in the following Table.

Table 1 pH Dry Cells Relative Yields Initial Final (g/dl) Pepsidines* Pepsidine C Control 6.5 8.8 1.12 100 100 with Precursor 6.5 9.1 1.20 112 114 A mixture consisting of Pepsidines.

absorption of the buffer solution which does not contain said cultivated broth is also measured as control. The balance between the two photo-absorption values thus obtained is taken as the value of the pepsin activity-inhibitory effect of the sample under test.

Independent of the abovementioned photo-absorption tests, a curve showing the pepsin inhibitory activity as the standard measure is graphically prepared using data of the photo-absorptions obtained in the similar manner as abovementioned with respect to the aqueous solutions .which contain the standard crystalline Pepsidine C in the different concentrations.

1 The total quantities of the Pepsidines contained in the cultivated broth are then obtained by comparative computation of the values of the pepsin-inhibitory activity relative to the abovementioned curve as the standard measure.

According to the present invention, N-acylpentapeptides, as will be obvious from the results obtained in the following working Examples, are obtained in higher yields than those obtained by the known arts. Furthermore, it is particularly notable that a marked enhancement in the yield of an intended specific N-pentapeptide can be attained by a selected use of the precursor.

Following Examples will serve to illustrate the specific embodiments of the invention without limitation.

EXAMPLE l 15 Liters of a nutrient medium containing 3% of glucose, 3% of peptone, 1% of meat extract and 0.5% of sodium chloride were prepared, which hereinafter called nutrient medium A. The nutrient medium A was sterilized in a 30 liter jar fermentor in accordance with a usual manner. After the sterilization with steam, the total volume of the sterilized nutrient medium amountedto 15.6 liters and had pH 6.5.

300 ml of a liquor of Streptomyces naniwaensis EF 44-201 strain separately cultivated were inoculated to the abovementioned sterilized nutrient medium, and the mixture was cultivated at 27C. for total 44 hours while agitating at 300 rpm. under aeration with 15 liters per minute of sterile air. After the cultivation for 21.5 hours, 500 ml of an aqueous solution which contained 150 grs. of sodium acetate were intermediately added to the cultivating liquor and the cultivation was further continued for the remaining period, the cultivated broth was filtered to remove the mycerium.

As for control, another cultivation was carried out under the same conditions as the above with exception The quantitative estimation of the individual Pepsidines thus obtained, on the other hand, is carried out as follows:

A butanol extract of the cultivated broth is spotted on a thin layer plate of silica gel and is developed with Benzene methanol acetic acid 20 5 by volume). The spots corresponding to the individual Pepsidines detected by means of Rydon-Smith reaction are separately recovered by scratching out from the plate and extracted with water. The pepsin-inhibitory activity of the individual Pepsidines is then measured in the manner as aforementioned.

As is evident from the data in the above Table, the relative yields of the Pepsidines were increased by the addition of sodium acetate as the precursor.

The filtrate was salted out with ammonium sulfate, and the recovered product was dissolved in methanol. The methanol solution was purified through a successive chromatography on active carbon and an anionexchange resin. There were obtained 3.9 grs. of Pepsidine C. In the similar manner, 3.2 grs. of Pepsidine C were obtained from the cultivated broth of the control.

EXAMPLE 2 l5 Liters of a nutrient medium hereinafter called nutrient medium B, were prepared which contained 5% of peptone, 0.1% of common salt, 0.1% of potassium dihydrogen phosphate, 0.000l% of ferrous sulfate, 0.000l% of manganese sulfate, 0.000l% of cupric sulfate and 0.0001% of zinc sulfate. To the nutrient medium B, there was added 1% of sodium acetate as the precursor.

The resulting nutrient medium was sterilized as usual in a 30 liter jar fermentator. After the sterilization, the total volume of the resulting nutrient medium was 14.5 liters and had pH 6.8.

As a control, another nutrient medium similar to the nutrient medium B was separately prepared without addition of sodium acetate.

To the two nutrient mediums thus prepared, there were inoculated Streptomyces naniwaensis EF 44-201 strain in the manner same as'that in Example 1.

After the 40 hours cultivations under aeration, the mycerin of the inoculated Streptomyces naniwaensis was removed from each of the cultivated liquor by filtration.

The data of the observations on the cultivated broths are shown as follows:

A mixture consisting of Pepsidines.

In comparison of the above data, it is noted that the considerable increments in the yields of Pepsidines, and especially Pepsidine C as compared with those of B at the time of the preparation. Cultivations and the determinations on the cultivated broth were carried out in accordance with those employed in Example 2. The

control, were obtained. 5 data of the observations are listed belows:

Table 4 Addition of Precursor Cultiv'n pH of Relative Yields Concn Time of Pepsi- Pepsidine C addition (hrs) Filtrate dines* S-Acetyl- 0 96 8.8 100 I00 glutathion ditto 0.] Beginning 96 8.9 152 184 AHMHA O 72 8.5 100 100 ditto 0.] Beginning 72 8.6 189 234 A mixture consisting of Pepsidines EXAMPLE 3 Each 100 ml of the nutrient mediums A and B re- I spectively prepared in the preceding Examples were devided into the shaking flasks of 50 ml capacity and the contents of the flasks were sterilized as usual. The sterilized mediums in the flasks were inoculated with Streptomyces naniwaensis EF 44-201 strain and the cultivations under shaking were continued at 27C.

Toward the end of 48 hours cultivation, there were added 1% of sodium acetate as the precursor to the cultivating medium A, and the cultivation was continued for further 24 hours.

To the nutrient medium B, on the other hand, 1% of sodium acetate was added at the commencement of cultivation and the cultivation was carried out continuously for 96 hours.

The data of the observations on the cultivated broths are shown as follows:

As is evident from the abovementioned data, the additions of the particular precursors considerably promote the yields of the Pepsidines as compared with those of the controls in which the precursors were absent. It is particularly noted that the effect coursed by the precursors is remarkable in the productivity of Pepsidine C.

What is claimed is:

1. In the method for providing N-acylpentapeptides by cultivation of the Streptomyces naniwaensis EF44- 201 (ATCC 21689) strain, the improvement wherein the culture is carried out in a nutrient medium containing a precursor of said N-acylpentapeptides selected from the group consisting of S-acetylglutathion, and 4-amino-3-hydroxy-6-methyl-heptanoic acid or derivatives thereof.

2 In the method as claimed in claim 1 wherein the N-acylpentapeptide is N-acetylpentapeptide.

Table 3 Cultivation Addit'n of precursor Relative Yields Time of pH of mehrs. addition Filtrate Pepsidines Pepsidine C dium after A 72 l 48 hrs. 8.5 123 123 B 96 0 8.9 I00 100 B 96 1 Beginning 8.9 153 193 A mixture consisting of Pepsidines.

respect to the cultivated broths to which the precursor was added.

EXAMPLE 4 0. l of S-acetylglutathion or AHMHA as the precursor was added to the abovementioned nutrient medium 3. In the method as claimed in claim I wherein the precursor is S-acetylglutathion.

4. In the method as claimed in claim 1 wherein the precursor is 4-amino-3-hydroxy-6-methyl-heptanoic acid. 

1. IN THE METHOD FOR PROVIDING N-ACYLPENTAPEPTIDES BY CULTIVATION OF THE STENPTOMYCES NANIWAENSIS EF44-201 (ATCC 21689) STRAIN, THE IMPROVEMENT WHEREIN THE CULTURE IS CARRIED OUT IN A NUTRIENT MEDIUM CONTAINING A PRECURSOR OF SAID N-ACYLPENTAPEPTIDES SELECTED FROM THE GROUP CONSISTING OF S-ACETYLGLUTATHION, AND 4-AMINO-3-HYDROXY-6-METHYL-HEPTANOIC ACID OR DERIVATIVES THEREOF.
 2. In the method as claimed in claim 1 wherein the N-acylpentapeptide is N-acetylpentapeptide.
 3. In the method as claimed in claim 1 wherein the precursor is S-acetylglutathion.
 4. In the method as claimed in claim 1 wherein the precursor is 4-amino-3-hydroxy-6-methyl-heptanoic acid. 